WO2020107548A1 - 高效酶催化合成血根碱与白屈菜红碱的方法 - Google Patents

高效酶催化合成血根碱与白屈菜红碱的方法 Download PDF

Info

Publication number
WO2020107548A1
WO2020107548A1 PCT/CN2018/121630 CN2018121630W WO2020107548A1 WO 2020107548 A1 WO2020107548 A1 WO 2020107548A1 CN 2018121630 W CN2018121630 W CN 2018121630W WO 2020107548 A1 WO2020107548 A1 WO 2020107548A1
Authority
WO
WIPO (PCT)
Prior art keywords
sanguinarine
gene
celandine
engineering bacteria
yeast engineering
Prior art date
Application number
PCT/CN2018/121630
Other languages
English (en)
French (fr)
Inventor
黄鹏
曾建国
Original Assignee
湖南美可达生物资源股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 湖南美可达生物资源股份有限公司 filed Critical 湖南美可达生物资源股份有限公司
Publication of WO2020107548A1 publication Critical patent/WO2020107548A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/03—Oxidoreductases acting on the CH-NH group of donors (1.5) with oxygen as acceptor (1.5.3)
    • C12Y105/03012—Dihydrobenzophenanthridine oxidase (1.5.3.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13055—Protopine 6-monooxygenase (1.14.13.55)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention relates to the technical field of catalytic synthesis of sanguinarine and celandine red alkali enzyme, in particular to a method for synthesizing sanguinarine and celandine red base by high-efficiency enzyme catalysis.
  • Bo Luo Hui Macleaya cordata (Willd.) R. Br. belongs to the poppy Cobo Luo Hui genus, also known as the trumpet pole, Luohui, mountain horn, mountain sycamore, Sanqiansan, etc., growing in hills and low mountains , Forest edge, grassland, roadside, is a wild herb.
  • Bo Luohui is mainly distributed in China, East Asia, North America and Europe.
  • Bo Luo Hui plants include Bo Luo Hui and Xiao Guo Bo Luo Hui (M.microcarpa (Maxim) Fedde) two species, as a traditional Chinese herbal medicine first seen in "Compendium of Materia Medica", in the folk as a maggot green herb widely used.
  • Boluhui has been found to have multiple pharmacological effects such as bacteriostasis, anti-inflammatory, and regulation of intestinal flora of livestock and poultry.
  • Boluhui began to be more and more widely used as a medicinal plant.
  • pro-opiodine and allobromine are precursors of sanguinarine and celandine, respectively, and pro-opioline in protropine-6-hydroxylase ( P6H) together with the coenzyme gene CPR to produce dihydrosanguine (DHSAN), then dihydrosanguinarine (DHSAN) to produce sanguinarine (SAN) under the catalysis of dihydrophenanthridine oxidase (DBOX) gene ; Alloinine is catalyzed by protropine-6-hydroxylase (P6H) and coenzyme gene CPR to produce dihydrocelandine (DHCHE), then dihydrocelandine (DHCHE) in two Under the catalysis of the hydrobenzophenanthridine oxidase (DBOX) gene, celandine (CHE) is produced.
  • P6H protropine-6-hydroxylase
  • DHCHE coenzyme gene CPR
  • Sanguinarine and celandine red alkali are the most important active ingredients in Bo Luohui extract, and the content in fruit pods is only about 0.5-2%.
  • the total alkaloid content in the leaves is about 50% of the pods, and the biological yield is more than double that of the pods.
  • the vast majority of Bo Luohui extracts are derived from the fruit pods of wild resources, with limited sources and low content of sanguinarine, resulting in the high price of sanguinarine, which limits the development of its industry.
  • the precursors which account for a large proportion of the total alkaloids, are not completely converted, and are treated as wastes in the extraction process without comprehensive and efficient use, resulting in great waste of resources.
  • Biotransformation also known as biocatalysis, refers to the use of microbial whole cells or extracted enzymes as catalysts to modify or directional synthesis of exogenous substrates to obtain valuable products of physiological and biochemical reactions, the essence of which is in biological systems Enzymatic reaction.
  • This particular enzyme-catalyzed reaction has the following characteristics: (1) It has a high degree of stereoselectivity. (2) Mild reaction conditions, safe production, no environmental pollution and simple post-processing. (3) Clear objectives, few by-products and low cost. (4) Microbial transformation can reduce the reaction steps.
  • the present invention intends to provide a method for efficiently synthesizing the enzyme sanguinarine and chelerythrine by yeast engineering bacteria.
  • the technical problem to be solved by the present invention is to provide an efficient enzyme-catalyzed method for synthesizing sanguinarine and celandine.
  • the present invention adopts the following technical solutions:
  • a method for catalyzing the synthesis of sanguinarine and celandine by high-efficiency enzyme which specifically includes the following steps:
  • Codon optimization of genes involved in the biosynthesis of sanguinarine and celandine red base first, according to the biosynthetic pathway of sanguinarine and celandine red base, from the known protropine-6-hydroxyl Heterologous expression (P6H), dihydrobenzophenanthridine oxidase (DBOX) genes and cytochrome P450 reductase (CPR) genes were screened for heterologous expression and result comparison analysis, respectively, to select the optimal genes with high expression efficiency. Then the codon optimization is performed on the selected optimal gene;
  • P6H protropine-6-hydroxyl Heterologous expression
  • DBOX dihydrobenzophenanthridine oxidase
  • CPR cytochrome P450 reductase
  • the optimized gene sequence is constructed on an expression vector, and then transferred into yeast engineering bacteria for transformation to obtain a recombinant yeast engineering strain;
  • step S3 Fermenting the leaf raw material solution of Bo Luohui with the recombinant yeast engineering bacteria constructed in step S3, and then collecting the cultured yeast engineering bacteria, lysing the bacterial cells, separating and purifying them, that is, obtaining sanguinarine and celandine.
  • the optimal gene in step S1 includes Protropine-6-hydroxylase (P6H) gene MC11229, and its nucleotide sequence is shown in SEQ ID No. 1.
  • the sequence of MC11229 after codon optimization is recorded as MC11229opt, which The nucleotide sequence is shown in SEQ ID No. 2.
  • the optimal gene in step S1 also includes the dihydrophenanthridine oxidase (DBOX) gene MC6408, whose nucleotide sequence is shown in SEQ ID No. 6; the codon-optimized sequence of MC6408 is recorded as MC6408opt, which The nucleotide sequence is shown in SEQ ID No. 7.
  • DBOX dihydrophenanthridine oxidase
  • the optimal gene in step S1 also includes the cucumber cytochrome P450 reductase gene CuCPR, whose nucleotide sequence is shown in SEQ ID No. 10.
  • Step S2 is as follows:
  • step S2 the plasmid of the expression vector is selected from PYES2.
  • step S2 the host strain of the yeast engineering strain is selected from yeast strain ivf.
  • step S3 the preparation method of the original solution of Bo Luohui blade is as follows:
  • step S3 are as follows:
  • the Bo Luohui leaf stock solution prepared by the above method is used as a substrate, and the precursor is fed to the yeast engineering bacteria constructed in step S2; at a temperature of 30°, the fermentation is cultured for 24 hours.
  • the functional gene MC11229 involved in the synthesis of sanguinarine and celandine red base in Boluohui is optimized according to the codons preferred by the yeast, and then the optimized sequence is integrated into S. cerevisiae.
  • Heterologous expression realizes the microbial transformation of sanguinarine and celandine, compared with the unoptimized functional genes, it can greatly improve the catalytic efficiency of the enzyme and increase the content of the catalytic products dihydrosanguinarine and sanguinarine , To reduce the production cost of dihydrosanguinarine and sanguinarine.
  • the functional gene MC6408 which participates in the synthesis of sanguinarine and celandine red base in Boluhui according to the codons preferred by the yeast, after codon optimization, then the obtained optimized sequence MC6408opt is integrated into S. cerevisiae Heterologous expression can achieve microbial transformation of sanguinarine and celandine, compared with unoptimized functional genes, it can greatly improve its enzyme catalytic efficiency and improve the catalytic products of dihydrosanguinarine and sanguinarine. Content, reduce the production cost of sanguinarine.
  • the Saccharomyces cerevisiae engineering bacteria constructed by the present invention which can efficiently transform pro-opioids and allobromine to form sanguinarine and celandine red base, is bio-transformed by using the powder of the leaves of Boluhui non-traditional medicinal parts as a substrate, At the same time, the fermentation conditions of the engineering bacteria were optimized to realize the comprehensive utilization of Boluhui resources, which can lay the foundation for reducing the production cost and industrial application of sanguinarine/celandine.
  • Bo Luo Hui Because the content of pro-opiodine and allobromine in the leaves of Bo Luo Hui is greater than that of sanguinarine and celandine, and the main effective components of Bo Luohui extract are sanguinarine and celandine.
  • the bio-transformation of Bo Luohui leaf raw material liquid is carried out directly, so that the original opioid and allobromine in the raw material are converted into high-value sanguinarine and celandine red base, which can improve the sanguinarine and celandine red.
  • the content of alkali can save the traditional operation of purifying pro-opiodine and allobromine, thereby reducing the production cost of sanguinarine and celandine, and can also realize the comprehensive utilization of Boluhui resources, which has a high Application value.
  • the present invention compares and screens out the optimal functional genes involved in the synthesis of sanguinarine and celandine, and performs codon optimization on the selected optimal functional genes to obtain gene optimization with higher enzyme catalytic efficiency Sequence to improve the content of sanguinarine and celandine from the genetic level; at the same time, it was constructed into yeast engineering bacteria, and the fermentation conditions of yeast engineering bacteria were studied and optimized to establish a high-yield blood root Standardized microbial fermentation process for alkali and celandine.
  • the raw material liquid of the non-traditional medicinal parts of Boluhui was directly fermented with engineering bacteria to convert the original opioids and allobromine in the leaves with high alkaloid content into High-value sanguinarine and celandine have high practical application value.
  • Figure 1 shows the synthetic route of Bohehuizhong sanguinarine and celandine
  • Fig. 2 is a graph showing the content of dihydrosanguinarine catalyzed by different P6H genes
  • Figure 3 shows the results of the determination of the content of dihydrosanguinarine catalyzed by the MC 11229 gene before and after optimization
  • Figure 4 is a graph showing the content of sanguinarine catalyzed by different DBOX genes
  • Figure 5 is a graph showing the results of the determination of the content of sanguinarine catalyzed by MC6408 before and after optimization;
  • Figure 6 is a graph showing the results of the determination of dihydrosanguinarine catalyzed by different CPR genes
  • Figure 8 is a comparison chart of the catalytic efficiency of different substrate concentrations
  • FIG. 10 is a comparison chart of the catalytic efficiency of fermenting and synthesizing celandine red base prepared by Bo Luohui leaf stock solution prepared under different pretreatment conditions and different recombinant yeast engineering bacteria constructed.
  • Protropine-6-hydroxylase is involved in sanguine (SAN) and celandine (CHE) A biosynthetic step that catalyzes the formation of allocryptine (ALL) to dihydrochelenine (DHCHE) and catalysis of pro-opiodine (PRO) to dihydrohematine (DHSAN).
  • ALL allocryptine
  • DHCHE dihydrochelenine
  • PRO pro-opiodine
  • DHSAN dihydrohematine
  • CPR cytochrome P450 reductase
  • the dihydrophenanthridine oxidase (DBOX) gene is involved in the biosynthesis steps of sanguinarine (SAN) and celandine (CHE), which can catalyze the production of chelone from dihydrocelandine (DHCHE) Betaine (CHE) and catalyzed dihydrosanguinarine (DHSAN) to produce sanguinarine (SAN).
  • SAN dihydrophenanthridine oxidase
  • CHE dihydrophenanthridine oxidase
  • DHCHE dihydrocelandine Betaine
  • DHSAN catalyzed dihydrosanguinarine
  • the present invention first compares the enzyme catalytic efficiency of P6H genes in poppy, poppy grass and Bo Luohui through the yeast expression system, and selects the optimal P6H gene, specifically combining the P6H gene of three species into a model plant and Arabidopsis (A .thaliana) CPR (AtCPR) gene was transferred into S. cerevisiae to construct yeast engineering bacteria. Then by feeding the substrate (original opioid standard), UPLC-QQQ MS quantitative analysis was used to compare the final product amount (dihydro sanguinarine) to compare the enzyme catalytic efficiency.
  • PsP6H is the P6H gene of Papaver somniferum (scientific name: Papaver somniferum L.), EcP6H is the P6H gene of E. sylvestris (scientific name: Eschscholtzia californica Cham.), and the genes PsP6H (GenBank KC154002), PsCPR (GenBank KF661328) are derived from NCBI, and the sequence information from NCBI, KBI661328 Synthesized by Jinweizhi Biological Technology Co., Ltd.
  • MC11229 and MC11218 are P6H genes of Bo Luohui, and their nucleotide sequences are shown in SEQ ID No. 1, SEQ ID No. 3, respectively.
  • the total RNA was extracted according to the polysaccharide polyphenol plant total RNA extraction kit, and it was reverse transcribed into cDNA using a reverse transcription kit.
  • the primer sequence table is shown in the following table 1-1:
  • PCR reaction system is calculated as 20 ⁇ l: 10-20ng/ ⁇ l template 1 ⁇ l, 10pmol/ ⁇ l forward and reverse primers 1 ⁇ l, 10mmol/L dNTP mix 0.4 ⁇ l, 0.5U/ ⁇ L high-fidelity Taq DNA polymerase 1 ⁇ l, 10 ⁇ 2 ⁇ l of PCR reaction buffer, the remainder is water.
  • PCR reaction conditions were: 94°C for 5 minutes; 94°C for 20 seconds, 55°C for 20 seconds, 72°C for 2 minutes and 30 seconds, 35 cycles; 72°C for 10 minutes.
  • reaction system is as follows 1-3:
  • the amplified fragment was ligated to a vector (Invitrogen) PYES2 with corresponding amino acid defects, and sequencing confirmed that there was no mutation.
  • the PYES2-Trp plasmid was transformed into yeast (ivf) strain alone to obtain yeast engineering strain MCY-3060; the recombinant plasmids PYES2-Ura+MC11218, PYES2-Ura+MC11229, PYES2-Ura+PsP6H, PYES2-Ura+EcP6H were respectively And PYES2-Leu+AtCPR were transferred into yeast (ivf) to obtain recombinant yeast engineering strains MCY-3061(PYES2+MC11218+AtCPR), MCY-3062(PYES2+MC11229+AtCPR), MCY-3063(PYES2+PsP6H+AtCPR ), MCY-3064 (PYES2+EcP6H+AtCPR).
  • Trp tryptophan
  • Leu leucine
  • Ura uracil
  • Induce yeast to express protein and then feed the precursor to collect the yeast. After lysis, extract the compound with methanol. After the sample is prepared, use UPLC-Q-TOF to detect it.
  • the measurement results are shown in Table 1-4 and Figure 2 below.
  • MCY-3060 is a yeast-engineered bacterium transferred into an empty vector as a blank control. MCY-3060 did not produce dihydrosanguinarine and sanguinarine after being fed pro-opioids under the same conditions, proving that yeast itself would not affect the experiment. Dihydrosanguinarine was detected in MCY-3061, MCY-3062, MCY-3063, MCY-3064, and the content was analyzed by SPSS 19.0 software, P ⁇ 0.05, the difference between the samples was significant, and the experimental results were statistically significant.
  • Select MC11229 (the nucleotide sequence of which is shown in SEQ ID No. 1) as the optimal gene. According to the codons preferred by Saccharomyces cerevisiae, after codon optimization, the gene optimized sequence MC11229opt is obtained, and its nucleotide sequence is as SEQ ID No. 2 shows.
  • the vector plasmids PYES2-Ura, PYES2-Leu, and PYES2-His were double digested with restriction enzymes.
  • the PYES2-Ura and PYES2-Leu double digestion reaction systems were the same as those in Table 1-2.
  • PYES2-His The double digestion reaction system is shown in the following table 1-6:
  • the amplified fragment was ligated to a vector (Invitrogen) PYES2 with corresponding amino acid defects, and sequencing confirmed that there was no mutation.
  • the gene sequence MC11229opt was constructed on the expression vector, and the recombinant expression vector was constructed according to the same method as the optimized gene sequence MC11229, and the recombinant expression vectors PYES2-His+MC11229opt, PYES2-Ura+MC11229 were obtained, and the recombination was also constructed.
  • the recombinant expression vectors PYES2-Ura+MC11229, PYES2-His+MC11229opt and PYES2-Leu+CuCPR were transferred into yeast (ivf, purchased from Thermo Fisher Scientific) to obtain yeast engineering strain MCY-3072 (PYES2-Ura+MC11229 +CuCPR)MCY-3083 (PYES2+MC11229opt+CuCPR), and also transfer the PYES2-Trp plasmid into the yeast (ivf) strain separately to obtain the yeast engineering strain MCY-3060 as a blank control; then separately in histidine (His) and Leu double defects and Trp, Leu and Ura three defects in SD/Dropout selection medium were cultured for 48h to obtain single colonies with a diameter of about lmm.
  • the present invention uses MCY-3060 as a blank control and does not produce dihydrosanguinarine and sanguinarine after feeding the original opioids under the same conditions, proving that yeast itself will not affect the experiment.
  • MCY-3072 and MCY-3083 both detected dihydrosanguinarine.
  • the content was analyzed by SPSS 19.0 software, P ⁇ 0.05, the difference between the samples was significant, and the experimental results were statistically significant.
  • the specific results are shown in Table 1-7:
  • Dihydrophenanthridine oxidase can both catalyze the production of chelerythrine (DHCHE) from dihydrocelandine (DHCHE) and DHSAN to produce sanguine (SAN) ).
  • the present invention takes PsDBOX, MC6408 and MC6407 genes as research targets, and transfers PsP6H and AtCPR into S. cerevisiae to construct yeast engineering bacteria, respectively.
  • UPLC-QQQ MS was used to quantitatively analyze the content of sanguinarine, and the enzyme catalytic efficiency of PsDBOX, MC6408 and MC6407 genes was compared.
  • PsDBOX is the DBOX gene of Papaver somniferum (scientific name: Papaver Somniferum L.).
  • the sequence information of PsDBOX is derived from NCBI and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • MC6408 and MC6407 are the P6H genes of Bo Luohui, and their nucleotide sequences are shown in SEQ ID No. 6 and SEQ ID No. 8, respectively. According to the polysaccharide and polyphenol plant total RNA extraction kit, extract the total RNA back and use reverse transcription kit to reverse transcribe it into cDNA.
  • Induce yeast to express protein and then feed the precursor to collect the yeast. After lysis, extract the compound with methanol. After the sample is prepared, use UPLC-Q-TOF to detect it.
  • the measurement results are shown in Table 2-2 and Figure 4 below.
  • MCY-3060 did not produce sanguinarine after being fed pro-opioids under the same conditions, proving that yeast itself would not affect the experiment. Both MCY-3065 and MCY-3066 detected sanguinarine.
  • the content was analyzed by SPSS 19.0 software, P ⁇ 0.05, the difference between the samples was significant, and the experimental results were statistically significant.
  • Select MC6408 (the nucleotide sequence of which is shown in SEQ ID No. 6) as the optimal gene. According to the codons preferred by Saccharomyces cerevisiae, after codon optimization, the gene optimized sequence MC 6408opt is obtained, and its nucleotide sequence is as SEQ ID No.7.
  • Recombinant expression vectors PYES2-Ura+MC6408, PYES2-His+MC6408opt and PYES2-Leu+CuCPR were transferred into yeast (ivf, purchased from Thermo Fisher Scientific) to obtain yeast engineering strain MCY-3084 (PYES2+MC11229+CuCPR +MC6408), MCY-3085 (PYES2+MC11229+CuCPR+MC6408opt), and also transfer the PYES2-Trp plasmid into yeast (ivf) strains separately to obtain the yeast engineering strain MCY-3060 as a blank control;
  • the histidine (His) and Leu double-deficient and Trp, Leu and Ura three-defective SD/Dropout selection medium were cultured for 48 hours to obtain single colonies with a diameter of about 1 mm.
  • Cytochrome P450 reductase cytochrome P450 reductase, CPR
  • CPR Cychrome P450 reductase
  • the electron donor NADPH electrons through flavin adenine dinucleotide (flavin adenosine dinucleotide (FAD) and flavin mononucleotide (FAD) flavin mononucleotide (FMN)
  • flavin adenine dinucleotide flavin adenosine dinucleotide (FAD) and flavin mononucleotide (FAD) flavin mononucleotide (FMN)
  • FAD flavin adenine dinucleotide
  • FAD flavin mononucleotide
  • FMN flavin mononucleotide
  • the present invention takes CuCPR, PsCPR, AtCPR, Mc19967 and Mc13802 genes as research targets, and reconstructs yeast engineering bacteria with MC11229. Then, by feeding the substrate, UPLC-QQQ MS quantitative analysis was used to compare the final product amount to compare the CPR enzyme catalytic efficiency.
  • CuCPR cucumber cytochrome P450 reductase (Cucumis sativus Linn.CPR) (provided by the cooperative team Huang Sanwen research team of Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences); PsCPR is poppy cytochrome P450 reductase; AtCPR is Arabidopsis cytochrome P450 Reductase; Mc19967 and Mc13802 are Bo Luohui cytochrome P450 reductase genes.
  • CuCPR cucumber cytochrome P450 reductase (Cucumis sativus Linn.CPR) (provided by the cooperative team Huang Sanwen research team of Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences); PsCPR is poppy cytochrome P450 reductase; AtCPR is Arabidopsis cytochrome P450 Reductase; Mc19967 and Mc13802 are Bo Luohui cytochrome P450 reductase genes.
  • the nucleotide sequences of CuCPR, PsCPR, AtCPR, Mc19967 and Mc13802 are shown in SEQ ID No. 10-14, respectively.
  • the sequence information of PsCPR and AtCPR comes from NCBI and is synthesized by Suzhou Jinweizhi Biological Technology Co., Ltd. According to the polysaccharide and polyphenol plant total RNA extraction kit, extract the total RNA back and use reverse transcription kit to reverse transcribe it into cDNA.
  • the primer sequence table is shown in the following table 3-1:
  • Recombinant plasmids PYES2-Leu+CuCPR, PYES2-Leu+PsCPR, PYES2-Leu+Mc19967, PYES2-Leu+Mc13802 and PYES2-Ura+MC11229 were transferred into yeast (ivf) to obtain yeast engineering strain MCY-3072 (PYES2 +MC11229+CuCPR), MCY-3073 (PYES2+MC11229+PsCPR), MCY-3074 (PYES2+MC11229+Mc19967), MCY-3075 (PYES2+MC11229+Mc13802), and then double-defective SD/ Leu and Ura Dropout selection medium was cultured for 48 hours to obtain single colonies with a diameter of about 1 mm.
  • MCY-3060 did not produce dihydro sanguinarine after being fed with pro-opioids under the same conditions, proving that yeast itself would not affect the experiment.
  • Dihydrosanguinarine was detected in MCY-3062, MCY-3072, MCY-3073, MCY-3074, MCY-3075, and the content results were analyzed by SPSS 19.0 software, P ⁇ 0.05, the difference between the samples was significant, and the experimental results were statistical Learn meaning.
  • the primer design of MC11229opt, MC6408opt, CuCPR refer to the above table 1-5, 2-3.
  • Recombinant plasmids PYES2-Ura+MC11229opt, PYES2-Leu+CuCPR, PYES2-Trp+MC6408opt were transferred into yeast (ivf) to obtain the best recombinant engineering strain MCY-3092 (PYES2+MC11229opt+CuCPR+MC6408opt).
  • the culture solution was centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded.
  • the content of pro-opiodine and allobromine in the leaves of Boluohui is greater than that of sanguinarine and celandine, and the main active ingredients of Boluohui extract are sanguinarine and celandine.
  • the bio-transformation of Bo Luohui leaf raw material liquid is carried out directly, so that the original opioid and allobromine in the raw material are converted into high-value sanguinarine and celandine red alkali, on the one hand, it improves the sanguinarine and celandine
  • the content of red alkali on the other hand, eliminates the traditional operation of purifying pro-opiodine and allobromine, thereby reducing the production cost of sanguinarine and celandine red alkali, and achieving the comprehensive utilization of Boluhui resources.
  • the TE buffer solution after the above different treatments was added under the same conditions, and the fermentation was cultured at a temperature of 30° for 24 hours; the cultured yeast engineering bacteria were collected, the cells were lysed, and the compounds were extracted with methanol to prepare samples.
  • MCY-3060 is used as a blank control, and the detected levels of sanguinarine and celandine can be used as the inherent content of sanguinarine and celandine in the TE buffer solution after adding leaf powder.
  • the specific results of sanguinarine and celandine after adding MCY-3092 engineering bacteria are shown in Table 4-3 and Table 4-4 and Figure 9 and Figure 10. After SPSS 19.0 software analysis, P ⁇ 0.05, the difference between the samples is significant, and the experimental results are statistically significant.
  • the above results show that: after adding engineering bacteria MCY-3092, the content of sanguinarine in the fermentation broth increased by about 3 times, and the content of celandine red alkali increased by about 2 times.
  • the results of different leaf material liquid pretreatment methods show that the content of sanguinarine and celandine red base obtained by ultrasonication of the leaf material liquid in an ultrasonic cleaner for 30 minutes is higher than that in a high-pressure steam sterilizer at 115°C for 30 minutes Content, and the catalytic efficiency of the engineered bacteria in the blade raw material liquid for 30 minutes of ultrasonication is also higher than that of high-pressure steam sterilization for 30 minutes.
  • the present invention compares and screens out the optimal functional genes involved in the synthesis of sanguinarine and celandine, and performs codon optimization on the selected optimal functional genes to obtain gene optimization with higher enzyme catalytic efficiency Sequence to improve the content of sanguinarine and celandine from the genetic level; at the same time, it was constructed into yeast engineering bacteria, and the fermentation conditions of yeast engineering bacteria were studied and optimized to establish a high-yield blood root Standardized microbial fermentation process for alkali and celandine. Finally, the raw material liquid of the non-traditional medicinal parts of Bo Luohui was directly fermented with engineering bacteria, and the pro-opioids and allobromine with high alkaloid content in the leaves were converted into high-value sanguinarine and celandine. Red alkali has high practical application value and realizes the comprehensive utilization of Boluhui resources.
  • SEQ ID No. 1-5 are the nucleotide sequences of MC11229, MC11229opt, MC11218, PsP6H, EcP6H respectively;
  • SEQ ID No. 6-9 are the nucleotide sequences of MC6408, MC6408opt, MC6407 and PsDBOX respectively;
  • SEQ ID 10-14 are the nucleotide sequences of CuCPR, PsCPR, AtCPR, Mc19967 and Mc13802 respectively;
  • SEQ ID No. 15-44 are primers PsP6H-Ura-F, PsP6H-Ura-R, EcP6H-Ura-F, EcP6H-Ura-R, MC11229-Ura-F, MC11229-Ura-R, MC11218-Ura- F, MC11218-Ura-R, AtCPR-Leu-F, AtCPR-Leu-R, YES2-Detect-F, YES2-Detect-R, MC11229opt-His-F, MC11229opt-His-R, CuCPR-Leu-F, CuCPR-Leu-R, MC6408-Trp-F, MC6408-Trp-R, MC6407-Trp-F, MC6407-Trp-R, PsDBOX-Trp-F, PsDBOX-Trp-R, MC6408opt

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

一种高效酶催化合成血根碱和白屈菜红碱的方法，其分别从已知的普罗托品-6-羟基化酶基因、二氢苯并菲啶氧化酶基因及细胞色素P450还原酶基因中通过异源表达和结果比对分析，筛选出表达效率高的最优基因，然后对选出的最优基因进行密码子优化；再将优化基因序列构建到表达载体上，之后转入酵母工程菌中进行转化获得重组酵母工程菌株；最后用博落回的叶片原料液前体饲喂重组酵母工程菌进行发酵，即得。本发明从基因水平、发酵工艺等多方面上提高血根碱和白屈菜红碱的酶催化效率，利用博落回的非传统药用部位叶片原料液直接与工程菌进行发酵，将叶片中生物碱含量高的原阿片碱和别隐品碱转化成高价值的血根碱和白屈菜红碱，以实现博落回资源的综合利用。

Description

高效酶催化合成血根碱与白屈菜红碱的方法 技术领域
本发明涉及血根碱和白屈菜红碱酶催化合成技术领域，具体涉及一种高效酶催化合成血根碱与白屈菜红碱的方法。
技术背景
博落回(Macleaya cordata(Willd.)R.Br.)属于罂粟科博落回属植物，别名又叫号筒杆、落回、山号筒、山梧桐、三钱三等，生长于丘陵、低山、林边、草地、路旁，是一种野生草本植物。博落回主要分布于中国、东亚、北美洲和欧洲。博落回属植物包括博落回和小果博落回(M.microcarpa(Maxim)Fedde)两个种，作为一种传统中草药最早见于《本草拾遗》，在民间作为一种杀蛆青草药而广泛使用。随着研究的不断深入，发现博落回具有抑菌、抗炎、调节畜禽类肠道菌群等多重药理作用，博落回开始作为一种药源植物得到越来越广泛的应用。
博落回中含有的血根碱、白屈菜红碱、原阿片碱和别隐品碱占博落回总生物碱的90％以上。现代药理研究表明原阿片碱、别隐品碱、白屈菜红碱和血根碱具有显著的生物活性，其中血根碱对治疗多种炎症有效，对畜禽类有较好的肠道菌群调节作用，目前已经作为饲用抗生素的替代品在欧洲等地区广泛销售。从2006年1月开始欧盟全面禁止在饲料中添加任何抗生素，导致血根碱的需求逐年增加。目前血根碱的主要来源是从博落回植株中提取，而博落回作为一种野生资源，这种获取方式导致其野生资源的存储量逐年减少。
现有的研究结果发现博落回的这4种主要生物碱在博落回中的分布呈现组织特异性。在成熟果荚中血根碱、白屈菜红碱占总碱的70％左右，原阿片碱和别隐品碱占30％左右；而在叶片中却相反，血根碱、白屈菜红碱占总碱的30％左右，原阿片碱和别隐品碱占70％左右。从生物合成途径上看(附图1)，原阿片碱和别隐品碱分别是血根碱和白屈菜红碱的前体物质，原阿片碱在普罗托品-6-羟基化酶(P6H)与辅酶基因CPR一起催化下生成二氢血根碱(DHSAN)，然后二氢血根碱(DHSAN)在二氢苯并菲啶氧化酶(DBOX)基因催化下生成血根碱(SAN)；别隐品碱在普罗托品-6-羟基化酶(P6H)与辅酶基因CPR一起催化下生成二氢白屈菜红碱(DHCHE)，然后二氢白屈菜红碱(DHCHE)在二氢苯并菲啶氧化酶(DBOX)基因催化下生成白屈菜红碱(CHE)。
血根碱和白屈菜红碱是博落回提取物中最主要的有效成分，在果荚中含量仅为0.5～2％左右。而叶片中的总生物碱含量大约是果荚中的50％左右，生物学产量是果荚的一倍以上。目前博落回提取物绝大部分来源于野生资源的果荚，来源有限，血根碱含量低，从而造成血根碱价格昂贵，而限制了其产业的发展。另一方面，占总生物碱很大比例的前体类物质未转 化完全，在提取过程中被当成废弃物未进行综合高效利用，导致资源的极大浪费。由于每提高0.1％植株中血根碱含量可降低10％的提取成本，而传统的栽培和育种进行改良所需周期长、受环境影响大、效率低、难以大规模种植和提升潜力有限，通过现代分子生物学技术体外构建工程化菌对前体物向终产物的有效转化是获得血根碱的新药源途径。
生物转化(biotransformation)也叫生物催化(biocatalysis)，是指利用微生物全细胞或提取酶作为催化剂对外源底物进行结构修饰或定向合成而获得有价值产物的生理生化反应，其本质是生物体系中酶的催化反应。这种特定的酶催化反应具有以下特点：(1)具有高度的立体选择性。(2)反应条件温和，生产安全，不造成环境污染和后处理简单。(3)目标明确，副产物少，成本低。(4)微生物转化可以减少反应步骤。
本发明拟提供一种酵母工程菌高效酶催化合成血根碱和白屈菜红碱的方法。
发明内容
本发明要解决的技术问题是提供一种高效酶催化合成血根碱和白屈菜红碱的方法。
为了解决上述技术问题，本发明采用以下技术方案：
提供一种高效酶催化合成血根碱和白屈菜红碱的方法，具体包括如下步骤：
S1、对参与血根碱和白屈菜红碱生物合成的基因进行密码子优化：首先根据血根碱和白屈菜红碱的生物合成途径，分别从已知的普罗托品-6-羟基化酶(P6H)、二氢苯并菲啶氧化酶(DBOX)基因及细胞色素P450还原酶(CPR)基因中通过异源表达和结果比对分析，分别筛选出表达效率高的最优基因，然后对筛选出的最优基因进行密码子优化；
S2、将优化后的基因序列构建到表达载体上，然后转入酵母工程菌中进行转化获得重组酵母工程菌株；
S3、将博落回的叶片原料液与步骤S3构建的重组酵母工程菌进行发酵，然后收集培养后的酵母工程菌，裂解菌体、分离纯化，即得血根碱和白屈菜红碱。
进一步地，
步骤S1中的最优基因包括普罗托品-6-羟基化酶(P6H)基因MC11229，其核苷酸序列如SEQ ID No.1所示，MC11229进行密码子优化后的序列记为MC11229opt，其核苷酸序列如SEQ ID No.2所示。
进一步地，
步骤S1中的最优基因还包括二氢苯并菲啶氧化酶(DBOX)基因MC6408，其核苷酸序列如SEQ ID No.6所示；MC6408进行密码子优化后的序列记为MC6408opt，其核苷酸序列如SEQ ID No.7所示。
进一步地，
步骤S1中的最优基因还包括黄瓜细胞色素P450还原酶基因CuCPR，其核苷酸序列如SEQ ID No.10所示。
进一步地，
步骤S2具体如下：
将博落回普罗托品-6-羟基化酶(P6H)基因优化序列MC11229opt与辅酶基因CuCPR、二氢苯并菲啶氧化酶(DBOX)基因优化序列MC6408opt一起构建到表达载体上，然后转入酵母工程菌中，并进行转化，获得重组酵母工程菌株。
进一步地，
步骤S2中，所述表达载体的质粒选自PYES2。
进一步地，
步骤S2中，所述酵母工程菌株的宿主菌选自酵母菌株ivf。
进一步地，
步骤S3中博落回叶片原液的制备方法如下：
(1)将博落回叶片放于35～45℃的恒温干燥箱中烘干，并粉碎得叶片粉末备用；
(2)然后将制得的叶片粉末按比例加入一定体积pH＝8.0的TE缓冲溶液中，配置呈一定比例的缓冲液；
(3)最后将所述缓冲液先放入高压蒸汽灭菌锅中110～120℃灭菌25～35min或者放入超声波清洗器中超声25～35min，然后4500～5500rpm离心4～6min，上清液过0.2～0.25μm滤膜，即得。
进一步地，步骤S3中的发酵条件具体如下：
将上述方法制得的博落回叶片原液作为底物，前体饲喂步骤S2构建的酵母工程菌；温度30°下，发酵培养24小时。
本发明的有益效果：
本发明将博落回中参与合成血根碱与白屈菜红碱的功能基因MC11229，根据酵母菌偏爱的密码子，进行密码子优化后，然后将得到的优化序列，整合到酿酒酵母中进行异源表达实现血根碱与白屈菜红碱的微生物转化，与未经优化的功能基因相比，能极大提高其酶催化效率，提高催化产物二氢血根碱及血根碱的含量，降低二氢血根碱及血根碱的生产成本。
本发明将博落回中参与合成血根碱与白屈菜红碱的功能基因MC6408，根据酵母菌偏爱的密码子，进行密码子优化后，然后将得到的优化序列MC6408opt，整合到酿酒酵母中进行异源表达实现血根碱与白屈菜红碱的微生物转化，与未经优化的功能基因相比，能极大提高其酶催化效率，提高催化产物二氢血根碱及血根碱的含量，降低血根碱的生产成本。
本发明构建的具有高效转化原阿片碱与别隐品碱生成血根碱和白屈菜红碱的酿酒酵母工程菌，以博落回非传统药用部位叶片的粉末为底物进行生物转化，同时还对工程菌的发酵条件进行了优化，实现博落回资源的综合利用，能为降低血根碱/白屈菜红碱的生产成本和工业化应用奠定基础。
由于，博落回叶片中原阿片碱与别隐品碱的含量大于血根碱与白屈菜红碱，而博落回提取物的主要有效成分是血根碱与白屈菜红碱。将博落回叶片原料液直接进行生物转化，使原料中的原阿片碱与别隐品碱转化成高价值的血根碱与白屈菜红碱，既可以提高血根碱与白屈菜红碱的含量，又可以省去传统提纯原阿片碱与别隐品碱的操作，从而降低血根碱和白屈菜红碱的生产成本，还可以实现博落回资源的综合利用，具有较高的应用价值。
综上，本发明从比较、筛选出参与合成血根碱与白屈菜红碱的最优功能基因，并且对筛选出的最优功能基因进行密码子优化，得到酶催化效率更高的基因优化序列，以期从基因水平上提高血根碱和白屈菜红碱的含量；同时将其构建到酵母工程菌中，并且对酵母工程菌的发酵条件进行了研究和优化，以期建立一个高产血根碱和白屈菜红碱的标准化微生物发酵工艺。最后，为实现博落回资源的综合利用，将博落回的非传统药用部位叶片原料液直接与工程菌进行发酵，将叶片中生物碱含量高的原阿片碱和别隐品碱转化成高价值的血根碱和白屈菜红碱具有较高的实际应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为博落回中血根碱和白屈菜红碱的合成途径；
图2为不同P6H基因催化产生二氢血根碱的含量结果图；
图3为优化前后的MC 11229基因催化产生二氢血根碱含量测定结果图；
图4为不同DBOX基因催化产生血根碱的含量结果图；
图5为优化前后的MC6408催化产生血根碱含量测定结果图；
图6为不同CPR基因催化产生二氢血根碱含量测定结果图；
图7为不同前体溶液催化效率比较图；
图8为不同底物浓度催化效率比较图；
图9为不同前处理条件下制得的博落回叶片原液与构建的不同重组酵母工程菌发酵培养合成血根碱的催化效率比较图；
图10为不同前处理条件下制得的博落回叶片原液与构建的不同重组酵母工程菌发酵培养合成白屈菜红碱的催化效率比较图。
具体实施方式
为了更好地阐述该发明的内容，下面通过具体实施例对本发明进一步的验证。特在此说明，实施例只是为更直接地描述本发明，它们只是本发明的一部分，不能对本发明构成任何限制。
如附图1的博落回中血根碱和白屈菜红碱的合成途径所示：普罗托品-6-羟基化酶(P6H)参与了血根碱(SAN)和白屈菜红碱(CHE)生物合成的步骤，其能催化别隐品碱(ALL)生成二氢白屈菜红碱(DHCHE)以及催化原阿片碱(PRO)生成二氢血根碱(DHSAN)。由于P6H属于细胞色素P450氧化还原酶，作为一种单加氧酶，需要与细胞色素P450还原酶(CPR)共表达才能发挥蛋白催化作用，CPR在反应中起到传递电子的作用。二氢苯并菲啶氧化酶(DBOX)基因参与了血根碱(SAN)和白屈菜红碱(CHE)生物合成的步骤，其能催化二氢白屈菜红碱(DHCHE)生成白屈菜红碱(CHE)以及催化二氢血根碱(DHSAN)生成血根碱(SAN)。
一、筛选最优基因并对最优基因进行密码子优化
(一)P6H最优基因的筛选与优化
在本申请人之前的研究中，在以罂粟和花菱草的P6H基因序列为参照，在博落回转录组数据中进行同源比对后找到2个同源性较高的基因序列(编号为MC11229和MC11218，参照专利：CN106119265A，博落回中参与血根碱与白屈菜红碱合成的细胞色素P450酶基因)，并进行了酵母异源表达验证。
本发明首先通过酵母表达系统比较罂粟、花菱草和博落回中P6H基因的酶催化效率，筛选出最优的P6H基因，具体是分别将三个物种的P6H基因组合模式植物及拟南芥(A.thaliana)中的CPR(AtCPR)基因转入酿酒酵母中构建酵母工程菌。再通过饲喂底物(原阿片碱标准品)的方式，利用UPLC-QQQ MS定量分析比较最终获得的产物量(二氢血根碱)来比较酶催化效率。
1、基因的获得
PsP6H为罂粟(学名：Papaver somniferumL.)的P6H基因，EcP6H为花菱草(学名：Eschscholtzia californica Cham.)的P6H基因，基因PsP6H(GenBank KC154002)、PsCPR(GenBank KF661328)序列信息来源于NCBI，由苏州金唯智生物科技有限公司合成。
MC11229、MC11218为博落回的P6H基因，其核苷酸序列分别如SEQ ID No.1、SEQ ID No.3所示。按照多糖多酚植物总RNA提取试剂盒提取博落回总RNA，并使用反转录 试剂盒将其反转录为cDNA。
2、然后利用正向引物和反向引物进行PCR扩增，引物序列表见下表1-1：
表1-1 引物序列表
Figure PCTCN2018121630-appb-000001
PCR反应体系以20μl计为：10-20ng/μl模板1μl，10pmol/μl正向、反向引物各1μl，10mmol/L dNTP mix 0.4μl，0.5U/μL高保真Taq DNA聚合酶1μl，10×PCR反应缓冲液2μl，余量为水。PCR反应条件为：94℃5分钟；94℃20秒，55℃20秒，72℃2分30秒，35个循环；72℃10分钟。
3、表达载体构建：用限制性内切酶KpnI-HF、XBaI将载体质粒PYES2-Ura、PYES2-Leu回收产物分别进行双酶切，反应体系如下小表1-2：
表1-2 双酶切反应体系
Figure PCTCN2018121630-appb-000002
Figure PCTCN2018121630-appb-000003
用限制性内切酶KpnI-HF、Sph-HF将载体质粒PYES2-Trp进行双酶切，反应体系如下1-3：
表1-3 PYES2-Trp双酶切反应体系
Figure PCTCN2018121630-appb-000004
将扩增得到的片段与具有对应氨基酸缺陷的载体(Invitrogen)PYES2连接，测序确认没有突变。
4、利用酵母表达系统验证上述各P6H基因的转化效率
将PYES2-Trp质粒单独转入酵母(ivf)菌株中，获得酵母工程菌株MCY-3060；将重组质粒PYES2-Ura+MC11218，PYES2-Ura+MC11229，PYES2-Ura+PsP6H，PYES2-Ura+EcP6H分别和PYES2-Leu+AtCPR转入酵母(ivf)中，获得重组酵母工程菌株MCY-3061(PYES2+MC11218+AtCPR)、MCY-3062(PYES2+MC11229+AtCPR)、MCY-3063(PYES2+PsP6H+AtCPR)、MCY-3064(PYES2+EcP6H+AtCPR)。然后再分别在色氨酸(Trp)单缺陷以及亮氨酸(Leu)与尿嘧啶(Ura)双缺陷的SD/Dropout选择培养基上培养48h，得到直径约l mm的单菌落。
诱导酵母表达蛋白，然后进行前体饲喂收集酵母，裂解后用甲醇抽提化合物,样品制备好后用UPLC-Q-TOF进行检测，测定结果如下表1-4及图2所示。
MCY-3060是转入空载体的酵母工程菌，作为空白对照。MCY-3060在相同条件下饲喂原阿片碱后未产生二氢血根碱和血根碱，证明酵母本身不会对实验产生影响。MCY-3061、MCY-3062、MCY-3063、MCY-3064均检测到了二氢血根碱，含量结果经SPSS 19.0软件分析，P＜0.05，样品之间差异显著，实验结果具有统计学意义。
表1-4 二氢血根碱含量测定结果
Figure PCTCN2018121630-appb-000005
Figure PCTCN2018121630-appb-000006
通过比较PsP6H、EcP6H、MC11229和MC11218基因的酶催化效率，我们发现MC11229+AtCPR工程菌催化产生了最高含量的二氢血根碱91.143±52.096ng﹒mL-1。且博落回的MC11229基因的催化效率是PsP6H基因的9.6倍，是EcP6H基因的5.7倍，同时是MC11218基因的9.6倍。
5、最优P6H基因MC11229的优化
选取MC11229(其核苷酸序列如SEQ ID No.1所示)作为最优基因，根据酿酒酵母偏爱的密码子，进行密码子优化后，得到基因优化序列MC11229opt，其核苷酸序列如SEQ ID No.2所示。
(1)制备博落回cDNA，然后利用正向引物和反向引物对基因进行PCR扩增，引物序列表见下表1-5：
表1-5 PCR引物序列与产物长度
Figure PCTCN2018121630-appb-000007
用限制性内切酶将载体质粒PYES2-Ura、PYES2-Leu、PYES2-His回收产物分别进行双酶切，PYES2-Ura、PYES2-Leu双酶切反应体系同表1-2，PYES2-His的双酶切反应体系见下表1-6：
表1-6 PYES2-His双酶切反应体系
Figure PCTCN2018121630-appb-000008
将扩增得到的片段与具有对应氨基酸缺陷的载体(Invitrogen)PYES2连接，测序确认没有突变。
将基因序列MC11229opt构建到表达载体上，并以优化前的基因序列MC11229为参照，按照同样的方法构建重组表达载体，获得重组表达载体PYES2-His+MC11229opt、PYES2-Ura+MC11229，同时还构建重组表达载体PYES2-Leu+CuCPR。
将重组表达载体PYES2-Ura+MC11229、PYES2-His+MC11229opt分别与PYES2-Leu+CuCPR转入酵母(ivf，购自Thermo Fisher Scientific公司)中，获得酵母工程菌株MCY-3072(PYES2-Ura+MC11229+CuCPR)MCY-3083(PYES2+MC11229opt+CuCPR)，同时还将PYES2-Trp质粒单独转入酵母(ivf)菌株中，获得酵母工程菌株MCY-3060，作为空白对照；然后再分别在组氨酸(His)与Leu双缺陷以及Trp、Leu与Ura三缺陷的SD/Dropout选择培养基上培养48h，得到直径约l mm的单菌落。
以pH＝8.0的TE缓冲溶液作为前体溶液，加入10μmol/L～2mmol/L原阿片碱作为底物，前体饲喂酵母工程菌；温度30°下发酵培养24小时。
收集培养后的酵母工程菌，裂解菌体、用甲醇抽提化合物，即得样品。将制备好的样品用UPLC-Q-TOF进行检测，结果如下表1-6和图3所示。
本发明以MCY-3060作为空白对照，在相同条件下饲喂原阿片碱后未产生二氢血根碱和血根碱，证明酵母本身不会对实验产生影响。MCY-3072、MCY-3083均检测到了二氢血根碱，含量结果经SPSS 19.0软件分析，P＜0.05，样品之间差异显著，实验结果具有统计学意义。具体结果见表1-7：
表1-7 优化前后的MC 11229基因二氢血根碱含量测定结果
Figure PCTCN2018121630-appb-000009
上表1-7的结果以及附图3表明，MC11229opt+CuCPR工程菌催化产生的二氢血根碱含量比MC11229+CuCPR的高，优化后的基因MC11229opt相比优化前的基因MC11229使得催化产物的含量得到了提高。具体地，优化后的酵母工程菌MC11229opt+CuCPR使得二氢血根碱的含量从20.096ng﹒mL-1提高到了26.944ng﹒mL-1，提高了34％。
(二)DBOX最优基因的筛选与优化
二氢苯并菲啶氧化酶(DBOX)既可以催化二氢白屈菜红碱(DHCHE)生成白屈菜红碱(CHE)又可以催化二氢血根碱(DHSAN)生成血根碱(SAN)。本发明以PsDBOX、MC6408和MC6407基因为研究目标，分别与PsP6H和AtCPR转入酿酒酵母中构建酵母工程菌。再以原阿片碱为底物，与工程菌共同进行发酵培养后，利用UPLC-QQQ MS定量分析血根碱的含量，比较PsDBOX、MC6408和MC6407基因的酶催化效率。
1、基因的获得
PsDBOX为罂粟(学名：Papaver somniferumL.)的DBOX基因，PsDBOX序列信息来源于NCBI，由苏州金唯智生物科技有限公司合成。
MC6408、MC6407为博落回的P6H基因，其核苷酸序列分别如SEQ ID No.6、SEQ ID No.8所示。按照多糖多酚植物总RNA提取试剂盒提取博落回总RNA，并使用反转录试剂盒将其反转录为cDNA。
2、然后利用正向引物和反向引物进行PCR扩增，引物序列表见下表2-1：
表2-1 PCR引物序列与产物长度
Figure PCTCN2018121630-appb-000010
Figure PCTCN2018121630-appb-000011
3、参照上述P6H最优基因的筛选与优化构建重组表达载体：
4、将重组质粒PYES2-Trp+PsDBOX，PYES2-Trp+MC6408，PYES2-Trp+MC6407分别和PYES2-Ura+PsP6H与PYES2-Leu+AtCPR转入酵母(ivf)中，获得重组酵母工程菌株MCY-3065(PYES2+PsP6H+AtCPR+MC6407)、MCY-3066(PYES2+PsP6H+AtCPR+MC6408)、MCY-30667(PYES2+PsP6H+AtCPR+PsDBOX)。然后再在Trp、Leu与Ura三个缺陷的SD/Dropout选择培养基上培养48h，得到直径约l mm的单菌落。
诱导酵母表达蛋白，然后进行前体饲喂收集酵母,裂解后用甲醇抽提化合物,样品制备好后用UPLC-Q-TOF进行检测，测定结果如下表2-2及图4所示。
以MCY-3060作为空白对照，MCY-3060在相同条件下饲喂原阿片碱后未产生血根碱，证明酵母本身不会对实验产生影响。MCY-3065和MCY-3066均检测到了血根碱，含量结果经SPSS 19.0软件分析，P＜0.05，样品之间差异显著，实验结果具有统计学意义。
表2-2 血根碱含量测定结果
Figure PCTCN2018121630-appb-000012
上述结果表明：工程菌PsP6H+AtCPR+MC6408催化产生的血根碱含量最高，其计算得出博落回的MC6408基因的酶催化效率最高。
5、最优DBOX基因MC6408的优化
选取MC6408(其核苷酸序列如SEQ ID No.6所示)作为最优基因，根据酿酒酵母偏爱的密码子，进行密码子优化后，得到基因优化序列MC 6408opt，其核苷酸序列如SEQ ID No.7所示。
(1)制备博落回cDNA，然后利用正向引物和反向引物对基因进行PCR扩增，引物序列表见下表2-3：
表2-3 PCR引物序列与产物长度
Figure PCTCN2018121630-appb-000013
3、参照上述P6H最优基因的筛选与优化构建重组表达载体。
4、将基因序列MC6408opt构建到表达载体上，并以优化前的基因序列MC6408为参照，按照同样的方法构建重组表达载体，获得重组质粒PYES2-Trp+MC6408、PYES2-Trp+MC6408opt，同时构建重组质粒PYES2-Leu+CuCPR、PYES2-Ura+MC11229；
将重组表达载体PYES2-Ura+MC6408、PYES2-His+MC6408opt分别与PYES2-Leu+CuCPR转入酵母(ivf，购自Thermo Fisher Scientific公司)中，获得酵母工程菌株MCY-3084(PYES2+MC11229+CuCPR+MC6408)、MCY-3085(PYES2+MC11229+CuCPR+MC6408opt)，同时还将PYES2-Trp质粒单独转入酵母(ivf)菌株中，获得酵母工程菌株MCY-3060，作为空白对照；然后再分别在组氨酸(His)与Leu双缺陷以及Trp、Leu与Ura三缺陷的SD/Dropout选择培养基上培养48h，得到直径约l mm的单菌落。
以pH＝8.0的TE缓冲溶液作为前体溶液，加入10μmol/L～2mmol/L原阿片碱作为底物，前体饲喂酵母工程菌；温度30°下发酵培养24小时。收集培养后的酵母工程菌，裂解菌体、用甲醇抽提化合物，即得样品。
将制备好的样品用UPLC-Q-TOF进行检测，结果如下表2-3和图5所示：
以MCY-3060作为空白对照，在相同条件下饲喂原阿片碱后未产生二氢血根碱和血根碱，证明酵母本身不会对实验产生影响。MCY-3084、MCY-3085均检测到了血根碱，含量结果经 SPSS 19.0软件分析，P＜0.05，样品之间差异显著，实验结果具有统计学意义。具体结果见表2-3：
表2-3 优化前后的MC6408基因血根碱含量测定结果
Figure PCTCN2018121630-appb-000014
上表2-3的结果以及附图5表明，工程菌MC11229+CuCPR+MC6408opt催化产生的血根碱含量比MC11229+CuCPR+MC6408高，优化后的基因MC6408opt相比优化前的基因MC6408使得催化产物的含量得到了提高。具体地，优化后的MC11229+CuCPR+MC6408opt酵母工程菌使得血根碱的含量从51.770ng﹒mL-1提高到了56.361ng﹒mL-1，提高了8.9％。
(三)最优CPR基因的筛选
细胞色素P450还原酶(cytochrome P450reductase,CPR)作为P450s电子传递链的重要功能单位，将电子供体NADPH的电子经过黄素腺嘌呤二核苷酸(flavin adenosine dinucleotide,FAD)和黄素单核苷酸(flavin mononucleotide,FMN)2个辅基传递给P450s，在P450s介导的氧化还原反应中起限速作用，是氧化还原反应中的关键酶。找到能在酵母表达系统中高效表达的CPR基因对于提高血根碱的含量具有非常重要的意义。在本申请人之前的研究中在博落回转录组数据中找到2个CPR的基因序列(编号为MC19967和MC13802)，并且进行了酵母异源表达验证。
本发明以CuCPR、PsCPR、AtCPR、Mc19967和Mc13802基因为研究目标，与MC11229重新构建酵母工程菌后。再通过饲喂底物的方式，利用UPLC-QQQ MS定量分析比较最终获得的产物量来比较CPR的酶催化效率。
1、基因的获得
CuCPR为黄瓜细胞色素P450还原酶(Cucumis sativus Linn.CPR)(由合作团队中国农业科学院蔬菜花卉研究所黄三文研究团队提供)；PsCPR为罂粟细胞色素P450还原酶；AtCPR为拟南芥细胞色素P450还原酶；Mc19967和Mc13802为博落回细胞色素P450还原酶基因。
CuCPR、PsCPR、AtCPR、Mc19967和Mc13802的核苷酸序列分别如SEQ ID No.10–14所示。PsCPR、AtCPR序列信息来源于NCBI，由苏州金唯智生物科技有限公司合成。按照多糖多酚植物总 RNA提取试剂盒提取博落回总RNA，并使用反转录试剂盒将其反转录为cDNA。
2、然后利用正向引物和反向引物进行PCR扩增，引物序列表见下表3-1：
表3-1 PCR引物序列与产物长度
Figure PCTCN2018121630-appb-000015
3、参照上述P6H最优基因的筛选与优化构建重组表达载体。
将重组质粒PYES2-Leu+CuCPR，PYES2-Leu+PsCPR，PYES2-Leu+Mc19967，PYES2-Leu+Mc13802分别与PYES2-Ura+MC11229转入酵母(ivf)中，获得酵母工程菌株MCY-3072(PYES2+MC11229+CuCPR)、MCY-3073(PYES2+MC11229+PsCPR)、MCY-3074(PYES2+MC11229+Mc19967)、MCY-3075(PYES2+MC11229+Mc13802)，然后再在Leu与Ura双缺陷的SD/Dropout选择培养基上培养48h，得到直径约l mm的单菌落。
以pH＝8.0的TE缓冲溶液作为前体溶液，加入10μmol/L～2mmol/L原阿片碱作为底物，前体饲喂酵母工程菌；温度30°下发酵培养24小时。收集培养后的酵母工程菌，裂解菌体、用甲醇抽提化合物，即得样品。
将制备好的样品用UPLC-Q-TOF进行检测，结果如下表3-2和图6所示。
MCY-3060作为空白对照，在相同条件下饲喂原阿片碱后未产生二氢血根碱，证明酵母本身不会对实验产生影响。MCY-3062、MCY-3072、MCY-3073、MCY-3074、MCY-3075均检测到了二氢血根碱，含量结果经SPSS 19.0软件分析，P＜0.05，样品之间差异显著，实验结果具有统计学意义。
表3-2 不同物种CPR催化生成二氢血根碱含量结果
Figure PCTCN2018121630-appb-000016
Figure PCTCN2018121630-appb-000017
上述结果显示，MCY3072工程菌催化产生的二氢血根碱94.194±24.981ng﹒mL-1是MCY3062的4.3倍，是MCY3074的3.7倍，是MCY3075的1.9倍，是MCY3073的1.7倍。即MC11229+CuCPR工程菌催化产生了最高含量的二氢血根碱，催化效率最好。这表明，在此试验条件下，CuCPR和MC11229在酵母中有高效表达。
(四)以上述(一)、(二)、(三)分别筛选和/或优化后的基因MC11229opt、MC6408opt、CuCPR构建最佳酵母工程菌合成血根碱和白屈菜红碱。
MC11229opt、MC6408opt、CuCPR的引物设计参照上表1-5,2-3.
参照上述P6H最优基因的筛选与优化构建重组表达载体，将重组质粒PYES2-Ura+MC11229opt、PYES2-Leu+CuCPR、PYES2-Trp+MC6408opt转入酵母(ivf)中，获得最佳重组酵母工程菌株MCY-3092(PYES2+MC11229opt+CuCPR+MC6408opt)。
1、配置不同的前体饲喂溶液，筛选最佳发酵条件
培养液5000rpm离心5min，弃上清。
(1)加入2mL含有终浓度分别为10μM、100μM、1mM、2mM原阿片碱、pH＝8.0的TE缓冲溶液。每个样品平行重复3份。30℃振荡培养16h。
(2)分别加入2mL含有终浓度为10μM原阿片碱、pH＝5.8和pH＝8.0的TE缓冲溶液和2mL含有终浓度为10μM原阿片碱、pH＝5.8和pH＝8.0的SD/Dropout缺陷型半乳糖液体培养基。每个样品平行重复3份。30℃振荡培养16h。
加入上述配置的不同pH的TE缓冲溶液和SD/Dropout缺陷型半乳糖液体培养基的相同浓度原阿片碱后，温度30°下，发酵培养24小时；收集培养后的酵母工程菌，裂解菌体、用甲醇抽提化合物，制得样品；检测到的血根碱具体结果见表4-1和图7。
同上，加入上述不同浓度原阿片碱pH＝8.0的TE缓冲溶液后，检测到血根碱的具体结果见表4-2和图8。经SPSS 19.0软件分析，P＜0.05，样品之间差异显著，实验结果具有统计学意义。
表4-1 不同前体溶液血根碱含量测定结果
Figure PCTCN2018121630-appb-000018
表4-2 不同底物浓度血根碱含量测定结果
Figure PCTCN2018121630-appb-000019
上述结果表明：以pH＝5.8和pH＝8.0的TE缓冲溶液为前体溶液时，在pH＝8.0的TE缓冲溶液中工程菌催化产生的血根碱含量较高；以pH＝5.8和pH＝8.0的SD/Dropout缺陷型半乳糖液体培养基作为前体溶液时，工程菌在pH＝8.0前体溶液中催化产生的血根碱含量较高。而在相同pH不同前体溶液的情况下，工程菌在TE缓冲溶液中催化产生的血根碱含量较高。因此我们可以得出工程菌在pH＝8.0的TE缓冲溶液中催化效率最高，是最佳的前体饲喂条件。
2、以博落回叶片原液饲喂上述构建的最佳酵母工程菌合成血根碱与白屈菜红碱。
博落回叶片中原阿片碱与别隐品碱的含量大于血根碱与白屈菜红碱，而博落回提取物的主要有效成分是血根碱与白屈菜红碱。将博落回叶片原料液直接进行生物转化，以使原料中的原阿片碱与别隐品碱转化成高价值的血根碱与白屈菜红碱，一方面提高血根碱与白屈菜红 碱的含量，另一方面省去传统提纯原阿片碱与别隐品碱的操作，从而降低血根碱和白屈菜红碱的生产成本，实现博落回资源的综合利用。
2.1博落回叶片原料液的前处理
(1)将博落回叶片放于40℃的恒温干燥箱中烘干，并用干磨机粉碎；
1)秤取0.5g粉末加入100mL pH＝8.0的TE缓冲溶液中；放入高压蒸汽灭菌锅中115℃灭菌30min。
2)秤取0.5g粉末加入100mL pH＝8.0的TE缓冲溶液中；放入超声波清洗器中超声30min。
(3)5000rpm离心5min，上清过0.22μm滤膜，备用。
2.2制备样品：菌株培养液5000rpm离心5min，弃上清；加入2mL 8.2.2中制备好的过膜上清液。每个样品平行重复3份，30℃振荡培养16h。
在相同条件下加入上述不同处理后的TE缓冲溶液，温度30°下，发酵培养24小时；收集培养后的酵母工程菌，裂解菌体、用甲醇抽提化合物，制得样品。MCY-3060作为空白对照，检测到的血根碱和白屈菜红碱含量可作为加入叶片粉末后的TE缓冲溶液中固有的血根碱和白屈菜红碱含量。加入MCY-3092工程菌后血根碱和白屈菜红碱的具体结果见表4-3、表4-4和图9、图10。经SPSS 19.0软件分析，P＜0.05，样品之间差异显著，实验结果具有统计学意义。
表4-3 植物组织发酵培养血根碱含量测定结果
编号 处理方式 血根碱含量(平均值±标准差)/ng﹒mL -1 转化率/％
MCY3060 TE灭菌 25.445±2.789 /
MCY3060 TE超声 31.205±0.784 /
MCY3092 TE灭菌 65.007±10.961 4.04
MCY3092 TE超声 85.415±11.887 6.40
表4-4 植物组织发酵培养白屈菜红碱含量测定结果
Figure PCTCN2018121630-appb-000020
上述结果表明：加入工程菌MCY-3092后，发酵液中血根碱的含量提高了约3倍，白屈菜红碱的含量提高了约2倍。不同叶片原料液前处理方式的结果表明，叶片原料液放入超声波清洗器中超声30min得到的血根碱和白屈菜红碱含量要高于在高压蒸汽灭菌锅中115℃灭菌30min的含量，并且工程菌在超声30min的叶片原料液中的催化效率也要高于高压蒸汽灭菌30min。
综上，本发明从比较、筛选出参与合成血根碱与白屈菜红碱的最优功能基因，并且对筛选出的最优功能基因进行密码子优化，得到酶催化效率更高的基因优化序列，以期从基因水平上提高血根碱和白屈菜红碱的含量；同时将其构建到酵母工程菌中，并且对酵母工程菌的发酵条件进行了研究和优化，以期建立一个高产血根碱和白屈菜红碱的标准化微生物发酵工艺。最后，将博落回的非传统药用部位叶片原料液直接与工程菌进行发酵，将叶片中生物碱含量高的原阿片碱和别隐品碱转化成高价值的血根碱和白屈菜红碱具有较高的实际应用价值，实现了博落回资源的综合利用。
以上所述为本发明的具体实施方式，但不能对本发明构成任何限制，因此需特别指出，凡是以本发明为基础，做得任何修改与改进均落在本发明保护范围之内。
序列说明：
SEQ ID No.1-5分别为MC11229、MC11229opt、MC11218、PsP6H、EcP6H的核苷酸序列；
SEQ ID No.6-9分别为MC6408、MC6408opt、MC6407以及PsDBOX的核苷酸序列；
SEQ ID10-14分别为CuCPR、PsCPR、AtCPR、Mc19967和Mc13802的核苷酸序列；
SEQ ID No.15-44分别为引物PsP6H-Ura-F、PsP6H-Ura-R、EcP6H-Ura-F、EcP6H-Ura-R、MC11229-Ura-F、MC11229-Ura-R、MC11218-Ura-F、MC11218-Ura-R、AtCPR-Leu-F、AtCPR-Leu-R、YES2-Detect-F、YES2-Detect-R、MC11229opt-His-F、MC11229opt-His-R、CuCPR-Leu-F、CuCPR-Leu-R、MC6408-Trp-F、MC6408-Trp-R、MC6407-Trp-F、MC6407-Trp-R、PsDBOX-Trp-F、PsDBOX-Trp-R、MC6408opt-Trp-F、MC6408opt-Trp-R、PsCPR-Leu-F、PsCPR-Leu-R、Mc19967-Leu-F、Mc19967-Leu-R、Mc13802-Leu-F、Mc13802-Leu-R的序列。

Claims (9)

  1. 一种高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，具体包括如下步骤：
    S1、对参与血根碱和白屈菜红碱生物合成的基因进行密码子优化：首先根据血根碱和白屈菜红碱的生物合成途径，分别从已知的普罗托品-6-羟基化酶基因、二氢苯并菲啶氧化酶基因及细胞色素P450还原酶基因中通过异源表达和结果比对分析，分别筛选出表达效率高的最优基因，然后对筛选出的最优基因进行密码子优化；
    S2、将优化后的基因序列构建到表达载体上，然后转入酵母工程菌中进行转化获得重组酵母工程菌株；
    S3、将博落回的叶片原料液与步骤S3构建的重组酵母工程菌进行发酵，然后收集培养后的酵母工程菌，裂解菌体、分离纯化，即得血根碱和白屈菜红碱。
  2. 根据权利要求1所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S1中的最优基因包括普罗托品-6-羟基化酶基因MC11229，其核苷酸序列如SEQ ID No.1所示，MC11229进行密码子优化后的序列记为MC11229opt，其核苷酸序列如SEQ ID No.2所示。
  3. 根据权利要求2所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S1中的最优基因还包括二氢苯并菲啶氧化酶基因MC6408，其核苷酸序列如SEQ ID No.6所示；MC6408进行密码子优化后的序列记为MC6408 opt，其核苷酸序列如SEQ ID No.7所示。
  4. 根据权利要求3所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S1中的最优基因还包括黄瓜细胞色素P450还原酶基因CuCPR，其核苷酸序列如SEQ ID No.10所示。
  5. 根据权利要求4所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S2具体如下：
    将博落回普罗托品-6-羟基化酶基因优化序列MC11229opt与辅酶基因CuCPR、二氢苯并菲啶氧化酶基因优化序列MC6408 opt一起构建到表达载体上，然后转入酵母工程菌中，并进行转化，获得重组酵母工程菌株。
  6. 根据权利要求5所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S2中，所述表达载体的质粒选自PYES2。
  7. 根据权利要求5所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S2中，所述酵母工程菌株的宿主菌选自酵母菌株ivf。
  8. 根据权利要求1-7任意一项所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S3中博落回叶片原液的制备方法如下：
    (1)将博落回叶片放于35～45℃的恒温干燥箱中烘干，并粉碎得叶片粉末备用；
    (2)然后将制得的叶片粉末按比例加入一定体积pH＝8.0的TE缓冲溶液中，配置呈一定比例的缓冲液；
    (3)最后将所述缓冲液先放入高压蒸汽灭菌锅中110～120℃灭菌25～35min或者放入超声波清洗器中超声25～35min，然后4500～5500rpm离心4～6min，上清液过0.2～0.25μm滤膜，即得。
  9. 根据权利要求1-7任意一项所述的高效酶催化合成血根碱和白屈菜红碱的方法，其特征在于，步骤S3中的发酵条件具体如下：
    将上述方法制得的博落回叶片原液作为底物，前体饲喂步骤S2构建的酵母工程菌；温度30°下，发酵培养24小时。
PCT/CN2018/121630 2018-11-27 2018-12-18 高效酶催化合成血根碱与白屈菜红碱的方法 WO2020107548A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811423227.1 2018-11-27
CN201811423227.1A CN109468351B (zh) 2018-11-27 2018-12-06 高效酶催化合成血根碱与白屈菜红碱的方法

Publications (1)

Publication Number Publication Date
WO2020107548A1 true WO2020107548A1 (zh) 2020-06-04

Family

ID=65674522

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/121630 WO2020107548A1 (zh) 2018-11-27 2018-12-18 高效酶催化合成血根碱与白屈菜红碱的方法

Country Status (2)

Country Link
CN (1) CN109468351B (zh)
WO (1) WO2020107548A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305855B (zh) * 2019-06-26 2022-03-22 昆明理工大学 天麻GeCPR基因及其应用
CN114517203A (zh) * 2022-03-18 2022-05-20 湖南美可达生物资源股份有限公司 一种博落回小檗碱桥酶基因优化序列及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105247038A (zh) * 2013-03-15 2016-01-13 小利兰·斯坦福大学托管委员会 产生苄基异喹啉生物碱(bia)的微生物及其制备和使用方法
CN106047904A (zh) * 2016-06-30 2016-10-26 湖南美可达生物资源有限公司 博落回中参与血根碱与白屈菜红碱合成的黄素蛋白氧化酶基因及其应用
CN106119265A (zh) * 2016-06-30 2016-11-16 湖南美可达生物资源有限公司 博落回中参与血根碱与白屈菜红碱合成的细胞色素p450酶基因及其应用
US20180163241A1 (en) * 2015-05-08 2018-06-14 The Board Of Trustees Of The Leland Stanford Junior University Methods of producing epimerases and benzylisoquinoline alkaloids
CN108837006A (zh) * 2018-07-24 2018-11-20 湖南美可达生物资源股份有限公司 一种博落回叶提取物及其制备方法与产品
CN109468350A (zh) * 2018-11-27 2019-03-15 湖南美可达生物资源股份有限公司 以博落回叶片原液为底物合成血根碱和白屈菜红碱的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105247038A (zh) * 2013-03-15 2016-01-13 小利兰·斯坦福大学托管委员会 产生苄基异喹啉生物碱(bia)的微生物及其制备和使用方法
US20180163241A1 (en) * 2015-05-08 2018-06-14 The Board Of Trustees Of The Leland Stanford Junior University Methods of producing epimerases and benzylisoquinoline alkaloids
CN106047904A (zh) * 2016-06-30 2016-10-26 湖南美可达生物资源有限公司 博落回中参与血根碱与白屈菜红碱合成的黄素蛋白氧化酶基因及其应用
CN106119265A (zh) * 2016-06-30 2016-11-16 湖南美可达生物资源有限公司 博落回中参与血根碱与白屈菜红碱合成的细胞色素p450酶基因及其应用
CN108837006A (zh) * 2018-07-24 2018-11-20 湖南美可达生物资源股份有限公司 一种博落回叶提取物及其制备方法与产品
CN109468350A (zh) * 2018-11-27 2019-03-15 湖南美可达生物资源股份有限公司 以博落回叶片原液为底物合成血根碱和白屈菜红碱的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 23 March 2015 (2015-03-23), "Cucumis Sativus NADPH--cytochrome P450 Reductase 2-like (LOC101219498), mRNA", XP55712561, Database accession no. XM_004146269 *
FOSSATI ELENA ET AL.: "Reconstitution of a 10- gene Pathway for Synthesis of the Plant Alkaloid Dihydrosanguinarine in Saccharomyces Cerevisiae", NATURE COMMUNICATION, vol. 5, 11 February 2014 (2014-02-11), XP055165144 *
LIU XIUBIN ET AL.: "The Genome of Medicinal Plant Macleaya Cordata Provides New Insights into Benzylisoquinoline Alkaloids Metabolism", MOLECULAR PLANT, vol. 10, no. 7, 5 July 2017 (2017-07-05), pages 977, XP55712548 *
TRENCHARD ISIS J. ET AL.: "Engineering Strategies for the Fermentative Production of Plant Alkaloids in Yeast", METABOLIC ENGINEERING, vol. 30, 14 May 2015 (2015-05-14), pages 97, XP055650686 *
ZENG JIANGUO ET AL.: "Integration of Transcriptome, Proteome and Metabolism Data Reveals the Alkaloids Biosynthesis in Macleaya Cordata and Macleaya Microcarpa", PLOS ONE, vol. 8, no. 1, 9 January 2013 (2013-01-09), pages 1, XP55712553 *

Also Published As

Publication number Publication date
CN109468351B (zh) 2020-07-14
CN109468351A (zh) 2019-03-15

Similar Documents

Publication Publication Date Title
Hernández et al. Saccharification of carbohydrates in microalgal biomass by physical, chemical and enzymatic pre-treatments as a previous step for bioethanol production
Pervez et al. Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process
Canam et al. Biological pretreatment with a cellobiose dehydrogenase-deficient strain of Trametes versicolor enhances the biofuel potential of canola straw
Xie et al. Simultaneous conversion of all cell wall components by an oleaginous fungus without chemi-physical pretreatment
KR101671435B1 (ko) 진세노사이드 생물전환능을 갖는 균주 및 이를 이용한 발효홍삼 추출물의 제조방법
CN113046248B (zh) 一株桔青霉菌xzh-16及其应用
WO2020107548A1 (zh) 高效酶催化合成血根碱与白屈菜红碱的方法
CN112126610A (zh) 一种用于生产羟基酪醇的工程菌
Li et al. Methane production from different parts of corn stover via a simple co-culture of an anaerobic fungus and methanogen
Tsuyama et al. Accumulation of sugar from pulp and xylitol from xylose by pyruvate decarboxylase-negative white-rot fungus Phlebia sp. MG-60
CN103937691B (zh) 一株产β‑果糖苷酶的米曲霉菌株及其培养方法与应用
Ilić et al. Valorization of lignocellulosic wastes for extracellular enzyme production by novel Basidiomycetes: screening, hydrolysis, and bioethanol production
TW200932912A (en) Biocarburant preparation using pencillium funiculosum enzymes
CN113512501B (zh) 一株草酸青霉菌xzh-2及其应用
Zhao et al. Influence of cellulase gene expression and cellulolytic activity on cellulose utilization in different Volvariella volvacea strains
CN108977367A (zh) 一株黑曲霉菌株及其在降解油茶饼粕中茶皂素的应用
CN102807958B (zh) 一种可分泌纤维素酶的菌株及其纤维素酶提取方法与应用
Xiao et al. Endophytic fungi from Dongxiang wild rice (Oryza rufipogon Griff.) show diverse catalytic potential for converting glycyrrhizin
CN109468350B (zh) 以博落回叶片原液为底物合成血根碱和白屈菜红碱的方法
CN109609522B (zh) 博落回二氢苯并菲啶氧化酶基因优化序列及其应用
CN104450769B (zh) 能提高达玛烯二醇转化效率的融合蛋白质及构建方法
Yuchun et al. Lignin degradation by Coprinus comatus in corn stalk
CN107746849A (zh) 一种甾体羟化酶基因的高效筛选方法
CN109609521B (zh) 博落回普罗托品-6-羟基化酶基因优化序列及其应用
CN117737029B (zh) 一种糖基转移酶突变体及其在络塞合成中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18941310

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18941310

Country of ref document: EP

Kind code of ref document: A1